This invention relates to the art of producing propagules from excised gymnosperm tissue. More particularly, this invention relates to an organogenetic method of in vitro clonal propagation of plantlets or propagules from excised gymnosperm tissue.
Approximately thirty species of gymnosperms, the so-called softwoods, comprise the great bulk of the commercially important timber species useful for construction lumber. Among these are the pines which include loblolly pine (Pinus taeda), slash pine (Pinus elliotii), longleaf pine (Pinus palustris), shortleaf pine (Pinus echinata), ponderosa pine (Pinus ponderosa), red pine (Pinus resinosa), jack pine (Pinus banksiana), Eastern white pine (Pinus strobus), Western white pine (Pinus monticola), sugar pine (Pinus lambertiana), lodgepole pine (Pinus contorta); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); the true firs including silver fir (Abies amabilis), grand fir (Abies grandis) noble fir (Abies procera), white fir (Abies concolor), balsam fir (Abies balsamea); and the cedars which include Western red cedar (Thuja plicata), incense cedar (Libocedrus decurrens), Port Orford cedar (Chamaecyparis lawsoniona), and Alaska yellow-cedar (Chamaecyparis nootkatensis), and Western larch (Laryx occidentalis).
Though not inclusive of all of the commercially important softwood species, the aforementioned group of conifers does include those pines which are generally considered to be commercially significant and which are or are becoming subject to intensive silvicultural management. Among these commercially significant pines, ponderosa pine, Western hemlock, Douglas-fir, and the four so-called southern yellow pines, slash, longleaf, shortleaf, and loblolly, are particularly important. Of this last group, loblolly pine and Douglas-fir have been the subject of intensive tree improvement breeding programs.
Loblolly pine, Pinus taeda, and Douglas-fir, Pseudotsuga menziesii, like many desirable species of trees, produce good seed crops only at infrequent and undependable intervals, and good cone crops typically occur only every five to seven years. In the normal course of events, a loblolly pine seedling produces male and female flowers when it is about 11 to 16 years old. When it does that, pollen from other trees will fertilize the female flowers, which will then produce seeds. About two years later, the seeds can be harvested and used to generate new plants. While the tree can pollinate some of its own female flowers so that some of the seedlings produced can be quite similar to the parent, none of the seedlings produced will be genetically identical.
Initially, the production of seedlings depended on wild seed which is drawn from an enormously varied gene pool. It was not long before foresters began to recognize that some seedlings grew far better in localized environments than others. For example, in the Douglas-fir region, it was found to be important to plant seedlings at the same approximate altitude from which the seed had been obtained. Soon it was realized that many other tree characteristics were heritable and while these traits vary from species to species, among them might be mentioned growth rates, the tendency to have straight or crooked stems, wood density, and light as opposed to heavy limbs. Nursery managers thus began searching their forests for and collecting seeds from wild trees that possessed one or more desirable characteristics. However, depending on the species, it may take from 15 to 50 years for a new generation to produce seeds of its own and several generations of breeding are required in order to maximize genetic improvement.
Accordingly, less time consuming methods have been sought to obtain genetically superior trees. To this end, vegetative propagation of pines, such as loblolly pine, by grafting or rooting of stem cuttings and needle fascicles has been carried out but these methods are inefficient and are not without their difficulties. Grafting is labor intensive and the percentage of success tends to be low because of graft or stock-scion incompatibility problems and, with rooting, the frequency of rooting tends to be very low and very young trees must generally be employed.
Accordingly, a goal of current research efforts is the development of reliable asexual methods for mass-producing identical copies of superior trees in great numbers. The advantages to be derived from such methods, in addition to the apparent economic advatages, include the potential for a drastic reduction in the time required to produce a second generation of trees from superior seedlings from a minimum of about 15 years to less than about 2 years.
Since a single cell in any plant or animal contains all of the genetic information necessary to replicate the entire organism, research efforts have tended in recent years to focus on the production of genetically superior seedlings by producing many plantlets from a single cluster of cells excised from a genetically superior tree, which method is commonly referred to in the art as clonal propagation or tissue-culture replication. The first tree reported to be produced by tissue-culture techniques was a quaking aspen at the Institute of Paper Chemistry in 1968. It has been discovered, however, that tissue-culture replication of conifers is not as easily accomplished.
Successful asexual clonal propagation techniques to date have been based on the biological processes known as organogenesis and embryogenesis. Organogenesis includes the initiation of shoots from meristematic centers induced in cultured tissue explants and the subsequent rooting of these shoots. As the method is generally employed, a portion of a donor plant is excised, sterilized and placed on a growth medium. The tissue most commonly used is a portion of young cotyledon from newly sprouted seeds or the intact embryo dissected from a seed. A much lower degree of success has been reported when tree leaves or stem tissues are cultured.
The growth medium consists of a basal nutrient medium of mineral salts and organic nutrients to which plant hormones have been added. One commonly used basal medium is that of Murashige and Skoog [Physiol. Plant. 15:473-497 (1962)]. This may be modified by addition of sucrose, myo-inositol, and thiamine [Cheng, T. Y., Plant Science Letters, 5:97-102 (1975)]. Various cytokinins and auxins are generally added to the basal medium to induce cell differentiation and growth. After an initial callus growth has formed which contains bud primordia, the plant material can be placed on a succession of different mediums that promote bud growth and shoot elongation. The shoot elongation medium may be free of hormones to reduce competing callus growth. The elongated shoots are excised and placed on a rooting medium consisting of the basal medium and generally with an auxin as the only exogenous hormone. When root primordia have formed, the shoots may be nourished on a medium free of added hormones in order to encourage root growth. The resultant plantlets are removed from the artificial media into a natural or synthetic soil mixture.
In the embryogenesis process, a group of cells become organized into a bipolar embryoid which will, in a favorable environment, develop bud primordia at one end and root primordia at the opposite end. One commonly reported route to production of plantlets by embryogenesis has been through suspension culture wherein groups of cells are suspended in a gently agitated liquid medium containing various plant growth hormones until bipolar embryoids are differentiated and developed. The embryoids are then placed on a nutrient medium for further development into plantlets.